Anthrax toxin is a three-part toxin secreted by Bacillus anthracis consisting of protective antigen (PA, 83 kDa), lethal factor (LF, 90 kDa) and edema factor (EF, 89 kDa) (Smith, H., et al., J. Gen. Microbiol., 29:517-521 (1962); Leppla, S. H., Sourcebook of bacterial protein toxins, p. 277-302 (1991); Leppla, S. H., Handb. Nat. Toxins, 8:543-572 (1995)), which are individually non-toxic. The mechanism by which individual toxin components interact to cause toxicity was recently reviewed (Leppla, S. H., Handb. Nat. Toxins, 8:543-572 (1995)). Protective antigen, recognized as central, receptor-binding component, binds to an unidentified receptor (Escuyer, V., et al., Infect. Immun., 59:3381-3386 (1991)) and is cleaved at the sequence RKKR167 (SEQ ID NO:1) by cell-surface furin or furin-like proteases (Klimpel, K. R., et al., Proc. Natl. Acad. Sci. USA, 89:10277-10281 (1992); Molloy, S. S., et al., J. B. Chem., 267:16396-16402 (1992)) into two fragments: PA63, a 63 kDa C-terminal fragment, which remains receptor-bound; and PA20, a 20 kDa N-terminal fragment, which is released into the medium (Klimpel, K. R., et al., Mol. Microbiol., 13:1094-1100 (1994)). Dissociation of PA20 allows PA63 to form heptamer (Milne, J. C., et al., J. Biol. Chem., 269:20607-20612 (1994); Benson, E. L., et al., Biochemistry, 37:3941-3948 (1998)) and also bind LF or EF (Leppla, S. H., et al., Bacterial protein toxins, p. 111-112 (1988)). The resulting hetero-oligomeric complex is internalized by endocytosis (Gordon, V. M., et al., Infect. Immun., 56:1066-1069 (1988)), and acidification of the vesicle causes insertion of the PA63 heptamer into the endosomal membrane to produce a channel through which LF or EF translocate to the cytosol (Friedlander, A. M., J. Biol. Chem., 261:7123-7126 (1986)), where LF and EF induce cytotoxic events.
Thus, the combination of PA+LF, named anthrax lethal toxin, kills animals (Beal, F. A., et al., J. Bacteriol., 83:1274-1280 (1962); Ezzell, J. W., et al., Infect. Immun., 45:761-767 (1984)) and certain cultured cells (Friedlander, A. M., J. Biol. Chem., 261:7123-7126 (1986); Hanna, P. C., et al., Mol. Biol. Cell., 3:1267-1277 (1992)), due to intracellular delivery and action of LF, recently proven to be a zinc-dependent metalloprotease that is known to cleave at least two targets, mitogen-activated protein kinase kinase 1 and 2 (Duesbery, N. S., et al., Science, 280:734-737 (1998); Vitale, G., et al., Biochem. Biophys. Res. Commun., 248:706-711 (1998)). The combination of PA+EF, named edema toxin, disables phagocytes and probably other cells, due to the intracellular adenylate cyclase activity of EF (Leppla, S. H., Proc. Natl. Acad. Sci. USA., 79:3162-3166 (1982)).
LF and EF have substantial sequence homology in amino acid (aa) 1-250 (Leppla, S. H., Handb. Nat. Toxins, 8:543-572 (1995)), and a mutagenesis study showed this region constitutes the PA-binding domain (Quinn, C. P., et al., J. Biol. Chem., 166:20124-20130 (1991)). Systematic deletion of LF fusion proteins containing the catalytic domain of Pseudomonas exotoxin A established that LF aa 1-254 (LFn) are sufficient to achieve translocation of “passenger” polypeptides to the cytosol of cells in a PA-dependent process (Arora, N., et al., J. Biol. Chem., 267:15542-15548 (1992); Arora, N., et al., J. Biol. Chem., 268:3334-3341 (1993)). A highly cytotoxic LFn fusion to the ADP-ribosylation domain of Pseudomonas exotoxin A, named FP59, has been developed (Arora, N., et al., J. Biol. Chem., 268:3334-3341 (1993)). When combined with PA, FP59 kills any cell type which contains receptors for PA by the mechanism of inhibition of initial protein synthesis through ADP ribosylating inactivation of elongation factor 2 (EF-2), whereas native LF is highly specific for macrophages (Leppla, S. H., Handb. Nat. Toxins, 8:543-572 (1995)). For this reason, FP59 is an example of a potent therapeutic agent when specifically delivered to the target cells with a target-specific PA.
The crystal structure of PA at 2.1 A was solved by X-ray diffraction (PDB accession 1ACC) (Petosa, C., et al., Nature, 385:833-838 (1997)). PA is a tall, flat molecule having four distinct domains that can be associated with functions previously defined by biochemical analysis. Domain 1 (aa 1-258) contains two tightly bound calcium ions, and a large flexible loop (aa 162-175) that includes the sequence RKKR167 (SEQ ID NO:1), which is cleaved by furin during proteolytic activation. Domain 2 (aa 259-487) contains several very long β-strands and forms the core of the membrane-inserted channel. It is also has a large flexible loop (aa 303-319) implicated in membrane insertion. Domain 3 (aa 488-595) has no known function. Domain 4 (aa 596-735) is loosely associated with the other domains and is involved in receptor binding. For cleavage at RKKR167 (SEQ ID NO:1) is absolutely required for the subsequent steps in toxin action, it would be of great interest to engineer it to the cleavage sequences of some disease-associated proteases, such as matrix metalloproteinases (MMPs) and proteases of the plasminogen activation system (e.g., t-PA, u-PA, etc., see, e.g., Romer et al., APMIS 107:120-127 (1999)), which are typically overexpressed in tumors.
MMPs and plasminogen activators are families of enzymes that play a leading role in both the normal turnover and pathological destruction of the extracellular matrix, including tissue remodeling (Birkedal-Hansen, H., Curr Opin Cell Biol, 7:728-735 (1995); Alexander, C. M., et al., Development, 122:1723-1736 (1996)), angiogenesis (Schnaper, H. W, et al., J Cell Physiol, 156:235-246 (1993); Brooks, P. C., et al., Cell, 92:391-400 (1998)), tumor invasion and metastasis formation. The members of the MMP family are multidomain, zinc-containing, neutral endopeptidases and include the collagenases, stromelysins, gelatinases, and membrane-type metalloproteinases (Birkedal-Hansen, H., Curr Opin Cell Biol, 7:728-735 (1995)). It has been well documented in recent years that MMPs and proteins of the plasminogen activation system, e.g., plasmiogen activator receptors and plasminogen activators, are overexpressed in a variety of tumor tissues and tumor cell lines and are highly correlated to the tumor invasion and metastasis (Crawford, H. C., et al., Invasion Metastasis, 14:234-245 (1995); Garbisa, S., et al., Cancer Res., 47:1523-1528 (1987); Himelstein, B. P., et al., Invest. Methods, 14:246-258 (1995); Juarez, J., et al., Int. J. Cancer, 55:10-18 (1993); Kohn, E. C., et al., Cancer Res., 55:1856-1862 (1995); Levy, A. T., et al., Cancer Res., 51:439-444 (1991); Mignatti, P., et al., Physiol. Rev., 73:161-195 (1993); Montgomery, A. M., et al., Cancer Res., 53:693-700 (1993); Stetler-Stevenson, W. G., et al., Annu Rev Cell Biol, 9:541-573 (1993); Stetler-Stevenson, W. G., Invest. Methods, 14:4664-4671 (1995); Davidson, B., et al., Gynecol. Oncol., 73:372-382 (1999); Webber, M. M., et al., Carcinogenesis, 20:1185-1192 (1999); Johansson, N., et al., Am J Pathol, 154:469-480 (1999); Ries, C., et al., Clin Cancer Res., 5:1115-1124 (1999); Zeng, Z. S., et al., Carcinogenesis, 20:749-755 (1999); Gokaslan, Z. L., et al., Clin Exp Metastasis, 16:721-728 (1998); Forsyth, P. A., et al., Br J Cancer, 79:1828-1835 (1999); Ozdemir, E., et al., J Urol, 161:1359-1363 (1999); Nomura, H., et al., Cancer. Res., 55:3263-3266 (1995); Okada, Y., et al., Proc. Natl. Acad. Sci. USA., 92:2730-2734 (1995); Sato, H., et al., Nature, 370:61-65 (1994); Chen, W. T., et al., Ann NY Acad Sci, 878:361-371 (1999); Sato, T., et al., Br J Cancer, 80:1137-43 (1999); Polette, M., et al., Int J Biochem cell Biol., 30:1195-1202 (1998); Kitagawa, Y., et al., J Urol., 160:1540-1545; Nakada, M., et al., Am J Pathol., 154:417-428 (1999); Sato, H., et al., Thromb Haemost, 78:497-500 (1997)).
Among the MMPs, MMP-2 (gelatinase A), MMP-9 (gelatinase B) and membrane-type 1 MMP (MT1-MMP) are reported to be most related to invasion and metastasis in various human cancers (Crawford, H. C., et al., Invasion Metastasis, 14:234-245 (1995); Garbisa, S., et al., Cancer Res., 47:1523-1528 (1987); Himelstein, B. P., et al., Invest. Methods, 14:246-258 (1995); Juarez, J., et al., Int. J. Cancer, 55:10-18 (1993); Kohn, E. C., et al., Cancer Res., 55:1856-1862 (1995); Levy, A. T., et al., Cancer Res., 51:439-444 (1991); Mignatti, P., et al., Physiol. Rev., 73:161-195 (1993); Montgomery, A. M., et al., Cancer Res., 53:693-700 (1993); Stetler-Stevenson, W. G., et al., Annu Rev Cell Biol, 9:541-573 (1993); Stetler-Stevenson, W. G., Invest. Methods, 14:4664-4671 (1995); Davidson, B., et al., Gynecol. Oncol., 73:372-382 (1999); Webber, M. M., et al., Carcinogenesis, 20:1185-1192 (1999); Johansson, N., et al., Am J Pathol, 154:469-480 (1999); Ries, C., et al., Clin Cancer Res., 5:1115-1124 (1999); Zeng, Z. S., et al., Carcinogenesis, 20:749-755 (1999); Gokaslan, Z. L., et al., Clin Exp Metastasis, 16:721-728 (1998); Forsyth, P. A., et al., Br J Cancer, 79:1828-1835 (1999); Ozdemir, E., et al., J Urol, 161:1359-1363 (1999); Nomura, H., et al., Cancer. Res., 55:3263-3266 (1995); Okada, Y., et al., Proc. Natl. Acad. Sci. USA., 92:2730-2734 (1995); Sato, H., et al., Nature, 370:61-65 (1994); Chen, W. T., et al., Ann NY Acad Sci, 878:361-371 (1999); Sato, T., et al., Br J Cancer, 80:1137-43 (1999); Polette, M., et al., Int J Biochem cell Biol., 30:1195-1202 (1998); Kitagawa, Y., et al., J Urol., 160:1540-1545; Nakada, M., et al., Am J Pathol., 154:417-428 (1999); Sato, H., et al., Thromb Haemost, 78:497-500 (1997)). The important role of MMPs during tumor invasion and metastasis is to break down tissue extracellular matrix and dissolution of epithelial and endothelial basement membranes, enabling tumor cells to invade through stroma and blood vessel walls at primary and secondary sites. MMPs also participate in tumor neoangiogenesis and are selectively upregulated in proliferating endothelial cells in tumor tissues (Schnaper, H. W, et al., J Cell Physiol, 156:235-246 (1993); Brooks, P. C., et al., Cell, 92:391-400 (1998); Chambers, A. F., et al., J Natl Cancer Inst, 89:1260-1270 (1997)). Furthermore, these proteases can contribute to the sustained growth of established tumor foci by the ectodomain cleavage of membrane-bound pro-forms of growth factors, releasing peptides that are mitogens for tumor cells and/or tumor vascular endothelial cells (Arribas, J., et al., J Biol Chem, 271:11376-11382 (1996); Suzuki, M., et al., J Biol Chem, 272:31730-31737 (1997)).
However, catalytic manifestations of MMP and plasminogen activators are highly regulated. For example, the MMPs are expressed as inactive zymogen forms and require activation before they can exert their proteolytic activities. The activation of MMP zymogens involves sequential proteolysis of N-terminal propeptide blocking the active site cleft, mediated by proteolytic mechanisms, often leading to an autoproteolytic event (Springman, E. B., et al., Proc Natl Acad Sci USA, 87:364-368 (1990); Murphy, G., et al., APMIS, 107:38-44 (1999)). Second, a family of proteins, the tissue inhibitors of metalloproteinases (TIMPs), are correspondingly widespread in tissue distribution and function as highly effective MMP inhibitors (Ki˜10−10 M) (Birkedal-Hansen, H., et al., Crit Rev Oral Biol Med, 4:197-250 (1993)). Though the activities of MMPs are tightly controlled, invading tumor cells that utilize the MMP's degradative capacity somehow circumvent these negative regulatory controls, but the mechanisms are not well understood.
The contributions of MMPs in tumor development and metastatic process lead to the development of novel therapies using synthetic inhibitors of MMPs (Brown, P. D., Adv Enzyme Regul, 35:293-301 (1995); Wojtowicz-Praga, S., et al., J Clin Oncol, 16:2150-2156 (1998); Drummond, A. H., et al., Ann NY Acad Sci, 30:228-235 (1999)). Among a multitude of synthetic inhibitors generated, Marimastat is already clinically employed in cancer treatment (Drummond, A. H., et al., Ann NY Acad Sci, 30:228-235 (1999)).
Here, as an alternate to the use of MMP inhibitors, we explored a novel strategy using modified PAs which could only be activated by MMPs or plasminogen activators to specially kill MMP- or and plasminogen activators-expressing tumor cells. PA mutants are constructed in which the furin recognition site is replaced by sequences susceptible to cleavage by MMPs or and plasminogen activators. When combined with LF or an LF fusion protein comprising the PA binding site, these PA mutants are specifically cleaved by cancer cells, exposing the LF binding site and translocating the LF or LF fusion protein into the cell, thereby specifically delivering a compounds, e.g., a therapeutic or diagnostic agent, to the cell.